Use of 2-(4-(4-chlorophenyl)cyclohexyl)-3-hydroxy-1,4-Naphthoquinone for the treatment of cancer

ABSTRACT

The present invention relates to the use of 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone or a physiologically acceptable salt or other physiologically functional derivative thereof for the manufacture of a medicament for the treatment of tumours in animals, to pharmaceutical compositions for the treatment of tumours in animals, to pharmaceutical compositions for the treatment of tumours, comprising said compound as active ingredient and to a method of treating tumours in an animal which comprises administering to said animal an effective amount of said compound.

This application is a 371 of PCT/9B93/01669 filed Aug. 6, 1993.

The present invention relates to the treatment of cancer. Moreparticularly, the invention is concerned with the use of2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone andphysiologically acceptable salts and physiologically functionalderivatives thereof as anti-tumour agents, and the use of said compoundfor the manufacture of medicaments for the treatment of cancer.

Research in the area of cancer chemotherapy has produced a variety ofanti-tumour agents, which have differing degrees of efficacy. Standardclinically used agents include adriamycin, actinomycin D, methotrexate,5-fluorouracil, cis-platinum, vincristine and vinblastine. However,these presently available anti-tumour agents are known to have variousdisadvantages, such as toxicity to healthy cells, and common sideeffects of anti-tumour drugs include alopecia and skin toxicity.

The compound 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone has previously been disclosed, for example inEuropean Patent No. 123,238 which relates to2-substituted-3-hydroxy-1,4-naphthoquinones of formula (I) ##STR1##wherein either R¹ is hydrogen and R² is selected from C₁₋₆ alkoxy,aralkoxy, C₁₋₆ alkyl-C₁₋₆ alkoxy, phenyl substituted by one or twogroups selected from halogen and C₁₋₆ alkyl, halogen and perhalo-C₁₋₆alkyl or R¹ and R² are both C₁₋₆ alkyl or phenyl, and n is zero or 1,and physiologically acceptable salts thereof. The compounds are said tohave antiprotozoal activity. Specifically, compounds of formula (I)wherein n is zero are said to be active against the human malariaparasite Plasmodium falciparum and also against Eimeria species such asE.tenella and E.acervulina, which are causative organisms of coccidiosisand compounds of formula (I) where n is 1 are said to be active againstprotozoa of the genus Theileria, in particular T.annulata or T.parva.Amongst the compounds specifically named and exemplified is the compoundof formula (I) wherein n is zero, R¹ is hydrogen and R² is4-chlorophenyl, i.e.2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone.

It has now surprisingly been found that2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone,represented in this specification by formula (II): ##STR2## exhibitsactivity against tumour cells, more specifically against carcinoma,adenocarcinoma and fibrosarcoma cells.

Thus, in a first aspect the present invention provides the use of thecompound of formula (II) and physiologically acceptable salts and otherphysiologically functional derivatives thereof for the manufacture of amedicament for the treatment of tumours in mammals (including humans).

According to a further aspect the present invention provides a methodfor the treatment of tumours in mammals, including humans whichcomprises the administration of an effective amount of a compound offormula (II), or a physiologically acceptable salt or otherphysiologically functional derivative thereof.

Treatment is particularly important for immunocompromised individuals,such as people with AIDS, who are more susceptible to certain types oftumour than people with a normally functioning immune system.

The hydroxyl group in the compound of formula (II) may form salts withappropriate bases, and physiologically acceptable salts of the compound(II) include inorganic base salts such as alkali metal (e.g. sodium andpotassium) salts and alkaline earth metal (e.g. calcium salts; organicbase salts e.g. phenylethylbenzylamine, dibenzylethylenediamine,ethanolamine and diethanolamine salts; and amino acid salts e.g. lysineand arginine.

Physiologically functional derivatives of formula (II) are derivativeswhich are converted in vivo to a compound of formula (II). Suchderivatives include those described in EPO362996 and EPO537947.

It will be appreciated that the compound of formula (II) may exist asthe cis or trans isomer, that is to say that the cyclohexyl ring may becis or trans substituted by the naphthoquinone nucleus and thechlorophenyl group. Both cis and trans isomers and mixtures thereof inany ratio may be used in accordance with the present invention. Ingeneral when the compound is in the form of a mixture of isomers thetrans isomer will be present in an amount of about 50% or will be thepredominant isomer but the use of mixtures in which the cis isomerpredominates is also included within the scope of the invention. Thespecific ratio of isomers may be varied as required; typical mixturesinclude those in which the cis/trans isomer ratio is about 1:1,40:60 and5:95. For use according to the present invention the trans isomer of thecompound of formula (II), or a mixture of its cis and trans isomerscontaining at least 95% e.g. 99% of the trans isomer, is preferred.

The compound of formula (II) may also exist in a tautomeric form inwhich the hydroxyl group donates its proton to one of the oxo groups andthe use of such tautomeric forms is included within the scope of thisinvention. However, it is believed that the stable form is that shown informula (II).

The amount of compound of formula (II) required to be effective as ananti-tumour agent will, of course, vary and is ultimately at thediscretion of the medical or veterinary practitioner. The factors to beconsidered include the route of administration and nature of theformulation, the mammal's bodyweight, age and general condition and thenature of the tumour to be treated. In general, a suitable effectiveanti-tumour dose for administration to man is in the range of 1.0 mg to200 mg per kilogram bodyweight per day, for example from 5 mg/kg to 100mg/kg, particularly 20 to 100 mg/kg.

It should be understood that the dosages referred to above arecalculated in terms of the compound of formula (II) per se.

For use according to the present invention the compound of formula (II)or a physiologically acceptable salt or other physiologically functionalderivative thereof is preferably presented as a pharmaceuticalformulation.

Pharmaceutical formulations comprise the active ingredient (that is, thecompound of formula (II) or a physiologically acceptable salt or otherphysiologically functional derivative thereof) together with one or morepharmaceutically acceptable carriers thereof and optionally othertherapeutic and/or prophylactic ingredients. The carrier(s) must beacceptable in the sense of being compatible with the other ingredientsof the formula and not deleterious to the recipient thereof.

The compound of formula (II) or its salt or other physiologicallyfunctional derivative may conveniently be presented as a pharmaceuticalformulation in unit dosage form. A convenient unit dose formulationcontains the active ingredient in an amount of from 10 mg to 3 g, e.g.50 mg to 3 g. A typical unit dose may contain for example 50 mg, 1 g, 2g or 3 g of the active ingredient.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal and parenteral(including subcutaneous, intradermal, intramuscular and intravenous),administration as well as administration by naso-gastric tube. Theformulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association the active ingredient with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of the active ingredient. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an inert liquid diluent. Tabletsmay be optionally coated and, if uncoated, may optionally be scored.Capsules may be prepared by filling the active ingredient either aloneor in admixture with one or more accessory ingredients, into the capsuleshells and then sealing them in the usual manner. Cachets are analogousto capsules wherein the active ingredient together with any accessoryingredient(s) is sealed in a rice paper envelope. The compound offormula (II) or a physiologically acceptable salt or otherphysiologically functional derivative thereof may also be formulated asdispersible granules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged e.g.in a sachet. Formulations suitable for oral administration wherein thecareer is a liquid may be presented as a solution or a suspension in anaqueous liquid or a non-aqueous liquid, or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms e.g. tablets wherein the active ingredient is formulated in anappropriate release--controlling matrix, or is coated with a suitablerelease--controlling film. Such formulations may be particularlyconvenient for prophylactic use.

The active ingredient my also be formulated as a solution or suspensionsuitable for administration via a naso-gastric tube.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping inmoulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of the active compound inaqueous or oleaginous vehicles. Injectible preparations may be adaptedfor bolus injection or continuous infusion. Such preparations areconveniently presented in unit dose or multi-dose containers which aresealed after introduction of the formulation until required for use.Alternatively, the active ingredient may be in powder form which isconstituted with a suitable vehicle, such as sterile, pyrogen-freewater, before use.

The compound of formula (II) or a physiologically acceptable salt orother physiologically functional derivative thereof may also beformulated as a long-acting depot preparation, which may be administeredby intramuscular injection or by implantation e.g. subcutaneously orintramuscularly. Depot preparations may include, for example, suitablepolymeric or hydrophobic materials, or ion-exchange resins. Suchlong-acting formulations are particularly convenient for prophylacticuse.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations for the various routes ofadministration described above may include, as appropriate one or moreadditional carrier ingredients such as diluents, buffers, flavouringagents, binders, surface active agents, thickeners, lubricants,preservatives (including anti-oxidants) and the like, and substancesincluded for the purpose of rendering the formulation isotonic with theblood of the intended recipient.

The compound of formula (II) or a physiologically acceptable salt orother physiologically functional derivative thereof may also be used inaccordance with the present invention in combination or concurrentlywith other therapeutic agents, for example agents used in the treatmentof immunocompromised patients, including antibacterial agents;antifungal agents; anticancer agents such as interferons e.g.alpha-interferon; antiviral agents such as azidothymidine(AZT,zidovudine); immunostimulants and immunodulators. The compound offormula (II) may also be administered in combination with a 4-pyridinolcompound, as described in EPA 123,239 e.g.3,5-dichloro2,6-dimethylpyridinol(meticlorpindol). The compound offormula (II) may also be administered in combination or concurrentlywith anti-diarrhoeal agents such as loperamide hydrochloride and/ordiphenoxylate hydrochloride, or with morphine sulphate. Oral rehydrationtherapy may also be carried out concurrently.

Compositions suitable for veterinary use include those adapted for oral,parenteral, and intrarumenal administration.

Compounds suitable for oral administration include drenches (oral liquiddosing), which may be solutions or suspensions; tablets, boluses,pastes, or in-feed preparations in the form of powders, granules orpellets.

Alternatively, veterinary compositions may be adapted to be administeredparenterally by sub-cutaneous, intramuscular or intravenous injection ofa sterile solution or suspension, by implantation or as an intramammaryinjection whereby a suspension or solution is introduced into the uddervia the teat.

For intrarumenal injection, the compositions of the invention may besolutions or solid or microcapusule suspensions. Typically thecompositions are similar to the oral liquid preparations or parenteralpreparations described herein. Such compositions are injected directlyinto the rumen, usually through the side of the animal, for example by ahypodermic syringe and needle or by an automatic injection devicecapable of giving single or multiple doses.

For veterinary administration the compound of formula (II) or its saltor other physiologically functional derivative is preferably formulatedwith one or more veterinarily acceptable carriers.

For oral administration, fine powders or granules may contain dilutingagents, for example lactose, calcium carbonate, calcium phosphate,mineral carriers, etc., dispersing and/or surface active agents, forexample polysorbates such as Tweens or Spans, and may be presented in adrench, in water or in a syrup, in a bolus, paste, or in a feedpreparation, in capsules or sachets in the dry state or in a non-aqueoussuspension, or in a suspension in water or syrup. Where desirable ornecessary, preserving, suspending, thickening or emulsifying agents canbe included. If intended for oral use, a bolus will be provided withretention means to inhibit regurgitation, for example it may be weightedwith a heavy density material such as iron or tungsten or the like ormay be retained by its shape, for example by wings which spring afteradministration. Boluses may contain disintegrating agents such as maizestarch or calcium or sodium methyl celluloses,hydroxypropylmethylcellulose, guar based vegetable gums, sodiumalginates or sodium starch glycolates; granulating or binding agentssuch as starch in the form of mucilage starch derivatives, such as "SnowFlake", cellulose derivatives such as talc, calcium stearate, methylcellulose, gelatin or polyvinylpyrrolidone; and/or lubricating agents,such as magnesium stearate or stearic acid. For parenteraladministration, the compounds may be presented in sterile injectionsolutions which may contain antioxidants or buffers, or as injectablesuspensions. Suitable solvents include water, in the case ofsuspensions, and organic solvents such as dimethylformamide,dimethylacetamide, diethylacetamide, ethyl lactate, ethyl akate,dimethylsulphoxide, alcohols, e.g. ethanol, glycols, e.g. ethyleneglycol, propylene glycol, butylene glycol and hexamethylene glycol,polyethylene glycols containing 2 to 159 ethylene glycol monomer unitsand having average molecular weights from about 90 to 7500, glycerinformal, glycofural, glycerol, isopropylmyristate N-methylpyrrolidone,2-pyrrolidone polyethylene glycoethers of tetrahydrofurfuryl alcohol anddiethylene glycol, and fixed and neutral oils, for example fractionatedcoconut oil. Parenteral formulations may also contain isotonic agents.

For veterinary use the compound of formula (II) may be employed togetherwith other therapeutic agents used in the field of animal health, forexample with anticoccidial and/or antitheilerial agents.

Methods for preparing the compound of formula (II) are described in EP123,238, and one specific method is illustrated in Example 1.

EXAMPLE 12-[trans-4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone

a) 4-(4-Chlorophenyl)cyclohexane-1-carboxylic Acid

Acetyl chloride (30 g) and finely powdered aluminium chloride (60 g)were stirred together in carbon disulphide (120 ml) and then cooled to-50° C., in a CO₂ /oxitol bath. Cyclohexene (30 g), previously cooled to-50° C., was added dropwise during 10 minutes while maintaining thetemperature of the reaction mixture at below -20° C. The mixture wasstirred at -50° C. for a further 60 minutes and the solvent thendecanted to leave a gummy orange complex. A little chlorobenzene wasadded as the material warmed to ambient temperature; the remainder ofthe chlorobenzene (total 300 ml) was then added, the so-obtainedsolution heated at 40° C. for 3 hours with stirring, poured onto amixture of ice and concentrated hydrochloric acid and the organic layerseparated, washed with 2M hydrochloric acid, 2M sodium hydroxide andwater, dried over anhydrous sodium sulphate and evaporated to dryness.The product was distilled in vacuo, the fraction boiling at 140°-154° C.(0.1 mm Hg) collected, diluted with an equal volume of petroleum ether(40-60), cooled to -6° C. and a continuous stream of nitrogen gasbubbled through, and the separated colourless solid recovered.

Bromine (2.8 ml) was added to a solution of sodium hydroxide (6.2 g) inwater (42 ml) at 0° C. The above-obtained substitutedhexahydroacetophenone (3.1 g) was dissolved in dioxan (15 ml) and thecold hypobromite solution then added, keeping the reaction mixture atbelow 20° C. The reaction mixture was stirred at ambient temperature for6 hours then allowed to stand overnight. Sodium metabisulphite was addedto destroy excess hypobromite, the mixture cooled and then acidified togive a colourless solid. The solid was filtered off, washed with water,dried and recrystallised from ethanol to give4-(4-chlorophenyl)cyclohex- ane-1-carboxylic acid, m.p. 254°-256° C.

b) 2-[4-(4-chlorophenyl)cyclohexyl]-3-chloro-1,4-naphthoquinone

A mixture of 2-chloro-1,4-naphthoquinone (3.95 g, 0.02 mol),4-(4-chlorophenyl)cyclohexane-1-carboxylic acid (4.9 g, 0.02 mol) andpowdered silver nitrate (1.05 g, 0.0062 mol) was heated to reflux withvigorous stirring in 40 ml of acetonitrile. A solution of ammoniumpersulphate (12.0 g, 0.0525 mol) in 50 ml of water was added dropwiseover 1 hour. The mixture was refluxed for 3 hours then cooled in ice for30 mins, after which it was filtered, and the residual sticky solidextracted twice with boiling chloroform to remove inorganic material.The chloroform was removed by evaporation to leave a yellow-brown solid(ca 2.7 g). This was dissolved in 40 nl of boiling acetonitrile; alittle insoluble material was removed by filtration. On cooling, thetitle compound separated as yellow crystals, (550 mg) m.p. 172°-175° C.

NMR, dH(d₆ -DMSO) 8.05(2H, mult., b-naphth), 7.85 (2H, mult., a-naphth),7.30 (4H, s., PhH), 3.30 (1H, br.t., CH), 2.67 (1H, br.t., CH), 1.2-2.4(8H, mult., 4×CH₂).

c) 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone

The product of stage (b) was suspended in 10 ml of boiling methanol and0.55 g of potassium hydroxide in 5.5 ml of water was added dropwise over15 mins. The mixture was refluxed until a dark red solution formed,(after ca. 6 hrs) when 2 ml of concentrated hydrochloric acid wascautiously added dropwise. The mixture was cooled and filtered, and thesolid residue washed thoroughly with water. The water washings werere-acidified and filtered. The combined solid residues (500 mg) mp200°-209°, were recrystallised from acetonitrile to give the titleproduct as the trans-isomer (300 mg) m.p. 216°-219° C.

EXAMPLE 2

The following examples illustrate, with reference to the compound offormula (II) per se, pharmaceutical and veterinary formulations whichmay be employed in accordance with the present invention.

A. Injectable solution

A solution for intramuscular injection may be prepared by mixing:

    ______________________________________                                        Compound of formula (II)                                                                          9.5    parts by weight                                    Dimethyl sulphoxide 19.0   parts by weight                                    Sorbitan monooleate 4.5    parts by weight                                    Corn oil            67.0   parts by weight                                                        100.0                                                     ______________________________________                                    

B. Injectable solution

The following injectable formulation was prepared:

    ______________________________________                                        Compound of formula (II)                                                                          5      parts by weight                                    N-methyl-pyroflidone                                                                              48.3   parts by weight                                    Tween 80            2      parts by weight                                    Span 80             4.7    parts by weight                                    Miglyol 812         40     parts by weight                                                        100.0                                                     ______________________________________                                    

C. Tablet

    ______________________________________                                        Compound of formula (II)                                                                              25.0   mg                                             Lactose BP              48.5   mg                                             Microcrystalline Cellulose BP                                                                         10.0   mg                                             ("Avicel pH 101")                                                             Low-substituted Hydroxypropyl;                                                                        10.0   mg                                             Cellulose BP ("LBPC LH-11")                                                   Sodium Starch Glycollate BP                                                                           3.0    mg                                             ("Explotab")                                                                  Povidone BP ("K30")     3.0    mg                                             Magnesium Stearate BP   0.5    mg                                                                     100.0  mg                                             ______________________________________                                    

D. Oral suspension

    ______________________________________                                        Compound of formula (II)                                                                             50 mg                                                  Avicel RC 591          75 mg                                                  Sucrose syrup          3.5 mg                                                 Methylhydroxybenzoate  5 mg                                                   Colour                 0.01% w/v                                              Cherry flavour         0.1% v/v                                               Tween 80               0.2% v/v                                               Water                  to 5 ml                                                ______________________________________                                    

E. Injectable suspension

    ______________________________________                                        Compound of formula (II)                                                                             100 mg                                                 Polyvinyl pyrrolidone (PVP)                                                                          170 mg                                                 Tween 80               0.2% v/v                                               Methylhydroxybenzoate  0.1% w/v                                               Water for Injection    to 3 ml                                                ______________________________________                                    

F. Capsule

    ______________________________________                                        Compound of formula (II) 100    mg                                            Starch 1500              150    mg                                            Magnesium stearate       2.5    mg                                            filled into a hard gelatin capsule                                            ______________________________________                                    

G. Aqueous Suspension

An aqueous suspension may be prepared as follows:

    ______________________________________                                        Compound of formula (II)                                                                         1.00    part by weight                                     Neosyl             16.00   parts by weight                                    Bentonite          3.20    parts by weight                                    Glycerin           15.00   parts by weight                                    Sodium benzoate    1.00    part by weight                                     Bevaloid 35/2      1.00    part by weight                                     Thymol             0.04    parts by weight                                    Water              62.76   parts by weight                                                       100.00                                                     ______________________________________                                    

H. Salt Block

A salt block may be prepared by mixing a finely divided compound offormula (II) (0.5 parts by weight) with sodium chloride (99.5 parts byweight) and the mixture pressed into blocks.

I. Paste

The following paste may be prepared:

    ______________________________________                                        Compound of formula (II)                                                                         3.0     parts by weight                                    Gum tragacanth     4.0     parts by weight                                    Bevaloid 35/3      1.0     part by weight                                     Nipagin "M"        0.1     parts by weight                                    Glycerin           19.0    parts by weight                                    Water              72.9    parts by weight                                                       100.00                                                     ______________________________________                                    

The use of the compound of formula (II) according to the presentinvention is illustrated by the following example:

BIOLOGICAL TEST RESULTS EXAMPLE 3 Activity Against Tumour Cell Lines InVitro

Test Compound

A: 2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone

Method

The activity of the test compound was evaluated using proliferativeassays against a number of different turnout cell lines in vitro.

The results, which are presented in Table 1 below, indicate that thetest compound was active, at 1001 μM concentration, against all the celllines tested.

                  TABLE 1                                                         ______________________________________                                                     Concentration                                                                             Activity                                             Cell Line    (μM)     (% Growth Inhibition)                                ______________________________________                                        P3888D1      100         92.26                                                (Mouse Leukaemia)                                                             DLD-1        100         94.18                                                (Human colon                                                                  adenocarcinoma)                                                               WiDr         100         79.44                                                (Human colon                                                                  adenocarcinoma)                                                               HCT-116      100         93.75                                                (Human colon                                                                  carcinoma)                                                                    A549         100         86.57                                                (Human lung                                                                   carcinoma)                                                                    ______________________________________                                    

EXAMPLE 4 Activity Against Human Fibrosarcoma Cells In Vitro

Test Compound

A: 2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone

Method

The activities of the test compound and of a soluble carbamatederivative thereof against two cell lines derived from humanfibrosarcoma cells were measured.

The results, which are presented in Table 2 below, show that both thecompound and the soluble derivative were active. The higher activity ofthe soluble derivative was almost certainly due to increased ease offormulation of the soluble derivative compared with the test compoundper se.

                  TABLE 2                                                         ______________________________________                                        IC.sub.50 (μM)                                                                           HT1080SCC2                                                                             HT10801C                                               ______________________________________                                        Test Compound   11         15                                                 Soluble Derivative                                                                            5.3        7.0                                                ______________________________________                                    

I claim:
 1. A method of treating a mammal having a carcinoma tumourwhich comprises administering to said mammal an effective carcinomatumour treatment amount of2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinine in theform of its trans isomer or a mixture of its cis and trans isomerscontaining at least 95% of the trans isomer or a physiologicallyacceptable salt thereof.
 2. The method of claim 1 wherein the amountadministered is 20 to 100 mg/kg of mammal bodyweight per day.
 3. Amethod of treating a mammal having an adenocarcinoma tumour whichcomprises administering to said mammal an effective adenocarcinomatumour treatment amount of2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinine in theform of its trans isomer or a mixture of its cis and trans isomerscontaining at least 95% of the trans isomer or a physiologicallyacceptable salt thereof.
 4. The method of claim 3 wherein the amountadministered is 20 mg to 100 mg/kg of mammal bodyweight per day.
 5. Amethod of treating a mammal having a fibrosarcoma tumour which comprisesadministering to said mammal an effective fibrosarcoma tumour treatmentamount of 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-l,4,-naphthoquininein the form of its trans isomer or a mixture of its cis and transisomers containing at least 95% of the trans isomer or a physiologicallyacceptable salt thereof.
 6. The method of claim 5 wherein theadministered is 20 to 100 mg/kg of mammal bodyweight per day.